PROJECT SUMMARY Embryonic cells polarize to develop specializations needed for morphogenesis and differentiation. Symmetrybreaking cues polarize many cells by altering RhoGTPase signaling, which triggers the asymmetric cortical localization of PAR polarity proteins. The molecular links that connect polarity cues, RhoGTPase signaling, and PAR asymmetries are poorly understood. Using the C. elegans embryo as a simple model, the long-term goal of this project is to determine how cell contact cues regulate RhoGTPases to induce the PAR asymmetries that polarize cells. Our ability to combine embryological manipulations with cell biological and genetic tools provides a unique opportunity to identify and characterize these mechanisms in living embryos. Given the deep conservation of cell polarity pathways and regulators, our studies will provide new insights into mechanisms of critical contact-mediated polarization events in humans. These include the polarization of embryonic blastomeres that occurs during compaction and is required for specification of the inner cell mass (which gives rise to the embryo proper and is the source of embryonic stem cells); and the polarization of epithelial cells that is needed for organogenesis and is essential for inhibiting tumor formation and invasion. In the prior award period, we defined a molecular pathway that mediates the contact-induced polarization of blastomeres by spatially altering the activity of the RhoGTPase CDC-42 a signaling protein with an ancient and broadly conserved role in cell polarity. We showed that cell contacts recruit the conserved RhoGAP protein PAC-1/ARHGAP10, which inactivates CDC-42 at contact sites. CDC-42 remains active at contact-free surfaces, where it recruits the PAR proteins PAR-3, PAR-6, and PKC-3/aPKC that then polarize each blastomere. In addition, we have obtained preliminary data suggesting that CDC-42 controls the localization of PAR-6 and PAR-3 through distinct mechanisms, and that CDC-42 localizes PAR-3 by regulating membrane trafficking. The goal of this proposal is to identify the molecular links between cell-cell contact, PAC-1 recruitment, and CDC-42 activity that lead to PAR-3 asymmetry. Our specific goals are (1) to determine how cell contacts between blastomeres recruit PAC-1 to locally inactivate CDC-42; (2) to identify the mechanisms that activate CDC-42 at contact-free surfaces; and (3) to test the hypothesis that CDC-42 controls PAR-3 asymmetry by regulating membrane trafficking. Our experiments will allow us to build a molecular pathway that connects cell-cell contact to CDC-42 activity and its regulation of polarity. We anticipate that our findings will provide general insights into the fundamental mechanisms of cell polarization, and specific molecular insights into how cell contacts polarize human blastomeres and epithelial cells to promote embryonic development and inhibit tumorigenesis